CN110029166A - Long-chain non-coding RNA LINC00205 is preparing diagnosis of ovarian cancer reagent or is treating the application in ovarian cancer - Google Patents
Long-chain non-coding RNA LINC00205 is preparing diagnosis of ovarian cancer reagent or is treating the application in ovarian cancer Download PDFInfo
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Abstract
MEHP processing significantly increases migration and the invasive ability of tumour cell, while MEHP can induce ovarian cancer cell that Epithelial and stromal conversion occurs as the result is shown；500nM concentration MEHP treated SKOV3 cell carries out transcript profile sequencing through the method for high-flux sequence and analyzes sequencing result, filters out 39 certified long-chain non-coding RNAs；In 39 lncRNA of differential expression, find that linc00205 Migration of Ovarian Cancer Cells caused by MEHP, invasive ability increase and Epithelial and stromal conversion has played certain effect by experimental verification.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to application field of the long-chain non-coding RNA for tumor suppression.
Background technique
Environment incretion interferent (Environmental endocrine disruptors, EEDs), also referred to as endocrine
Chaff interferent (Endocrine disrupting chemicals, EDCs) refers to maintenance organismic internal environment stable state and adjusts hair
Educate the generation of the internal natural hormone of process, release, transhipment, metabolism, combination, effect cause the one kind seriously affected naturally occurring
Or the exogenous material of pollution.It has been found or has hundreds of suspected of the Enviromental pollutants with endocrine, wrap
Include phthalate, polychlorinated biphenyl, organochlorine insecticide, induced by alkyl hydroxybenzene and bisphenol compound class etc..Usually by dry
The endocrine organ and tissue disturbed classifies, such as estrogen chaff interferent, androgen chaff interferent, Thyroid Hormone Disruptors, sugared skin
Matter hormone chaff interferent, growth hormone chaff interferent etc..It is now recognized that EDCs and dysgenesia, birth defect, dysplasia, metabolism are disorderly
The occurrence and development of random and certain cancers (such as breast cancer, carcinoma of testis, oophoroma) are related.People can lead in daily life
It crosses air pollution, pesticide, industrial chemicals, heavy metal and food and touches various EDCs, therefore EDCs is strong for human body
The influence of health is hot issue now.
The frequent species of phthalic ester plasticizer include: the own ester (DEHP) of phthalic acid two (2- ethyl), neighbour
Dicyclo-hexyl phthlate (DCHP), BBP(Butyl Benzyl Phthalate (BBP), phthalic acid diformazan (DMP) etc..DEHP is a kind of
Global annual output is up to three to four million tons of plasticisers most generally used[10][11], it is widely used in polyvinyl chloride plastic material products, such as
Medical supplies, polybag and toy.There are many kinds of the route of exposure of DEHP, and in all approach, DEHP mainly passes through and disappears
Change road, respiratory tract and skin contact to be absorbed into vivo, wherein alimentary canal contact is considered as the master of general population contact DEHP
Want mode, it was reported that the DEHP intake estimated daily is 3 to 30 mg/kg body weight/days.Due to DEHP and plastic polymer
Object is easy to immerse in environment not by covalent linkage.In 2011, international cancer research institution (IARC) was again
The classification of DEHP is had evaluated, and it is upgraded to 2B class carcinogen from 3 class carcinogens.Before research shows that mammal
Hydrolase present in enteron aisle can convert DEHP to its primary metabolite phthalic acid list ethylhexyl (MEHP),
MEHP has genotoxicity and development toxicity simultaneously, and toxicity is higher than DEHP.Epidemiological study prompts DEHP and MEHP in steroid
(mammary gland, uterus, testis, prostate) may play an important role in hormone-dependent type tumour.
Malignant tumor of ovary is common one of the malignant tumour of female sex organ[18](cervical carcinoma, oophoroma, intrauterine
Film cancer), disease incidence is only second to cervix cancer and carcinoma of endometrium.It is most common with epithelioma in malignant tumor of ovary, followed by dislike
Sexual reproductive cell tumour.Wherein ovarian epithelium mortality of carcinoma accounts for the first place of all kinds of gynecological tumors, causes serious prestige to women life
Side of body transfer and invasion are the lethal most important reasons of oophoroma.When oophoroma lesion is only confined in ovary, 5 years
Survival rate reaches 90% or more；And when lesion is transferred in peritonaeum, five year survival rate drops sharply to 20% or less[28]。
Early diagnosis before the transfer of ovary carcinogenesis is the key that improve therapeutic effect and five year survival rate, therefore understand ovary
The mechanism of carcinogenesis transfer is most important.
High-flux sequence (High-Throughput Sequencing), also known as next-generation sequencing (Next-Generation
Sequencing), compared with first generation sequencing approach, major advantage is to the numbers a greater amount of with lower cost output
According to；Compared with traditional DNA microarray, high-flux sequence can in the case where not needing specific probe identification and quantification it is dilute
There is transcript.High throughput sequencing technologies can be used for gene order-checking and resurvey sequence, transcriptome analysis (RNA-Seq), DNA protein
It interacts (ChIP-Seq) and apparent gene group is sequenced[31].Transcript profile (transcriptome) is referring broadly to a certain physiology
Under the conditions of, the set of intracellular all transcription products, including mRNA, rRNA, transfer RNA and non-coding RNA；It is narrow
Refer to the set of all mRNA in justice.For transcript profile research for illustrate genome function and disclose cell and tissue
Molecular composition ingredient and understanding development and disease are most important.The main purpose of transcription group research is: by all species
Transcript is catalogued, including mRNA, non-coding RNA and tiny RNA；It is repaired after initiation site, shear mode and other transcriptions
Decorations etc. determine the transcription structure of gene；And quantitative transcript expression changes during development and under varying environment
Become.
Phthalic acid list ethylhexyl (MEHP) is the metabolite of DEHP, and the LD50 of rat oral contact MEHP is
1340mg/kg, influence of the endocrine played to human health is equally current research hotspot.It need not set
It doubts, EDCs pollutes the great public health problem for having become global facing, and EDCs and human reproduction's health are that 21 century is raw
The important scientific problems that life science and environmental science are urgently studied and solved.Due to containing these chemicals in cosmetics
Matter, young women may be easier exposed to phthalate substance compared to the male of same age bracket, so people
Influence of the PAEs for female reproductive system should be increasingly focused on.
The present invention is based in the prior art for MEHP at low concentrations with the research of the relationship of oophoroma deficiency, respectively from
Multiple sides such as influence of the MEHP to abortion syndrome cell viability, migration and invasive ability and correlative protein expression level
Face to inquire into whether the MEHP in physiological concentration range can promote the development of human ovarian cancer, and further studies it and promotees tumour
Molecular mechanism.
Summary of the invention
It is an aspect of the invention to provide a kind of diagnosis targets of tumour poor prognosis；In a specific embodiment
In, the tumour is gynecological tumor, preferably oophoroma；In another specific embodiment, the poor prognosis is
As caused by environment incretion interferent, specifically, the environment incretion object is phthalic ester plasticizer, more
Body, the environment incretion object is the primary metabolite MEHP of DEHP in vivo；In another specific embodiment,
The target is non-coding RNA, and further, the non-coding RNA is long-chain non-coding RNA；Further, described
The high expression in the ovarian cancer cell line that MEHP is handled of long-chain non-coding RNA；Further, the long-chain non-coding
RNA is selected from linc01233, linc00205, MIR193BHG, GAS5 and NOP14-AS1, it is preferred that the non-volume of the long-chain
Code RNA is linc00205.In a specific embodiment, the nucleotides sequence of the linc00205 is classified as SEQ ID NO:
Shown in 1 or the nucleotides sequence of the linc00205 is classified as with sequence shown in SEQ ID NO:1 with 99% homology
And derive from the nucleic acid sequence of the mankind.
Another aspect of the present invention is that long-chain non-coding RNA is preparing the application in diagnostic reagent.It is specific at one
In embodiment, the diagnostic reagent is for diagnosis of ovarian cancer；In another specific embodiment, the long-chain is non-
Coding RNA high expression in the ovarian cancer cell line that MEHP is handled；Further, the long-chain non-coding RNA is selected from
Linc01233, linc00205, MIR193BHG, GAS5 and NOP14-AS1, it is preferred that the long-chain non-coding RNA is
linc00205.In a specific embodiment, the nucleotides sequence of the linc00205 is classified as shown in SEQ ID NO:1,
Or the nucleotides sequence of the linc00205 is classified as with sequence shown in SEQ ID NO:1 with 99% homology and source
In the nucleic acid sequence of the mankind.
Another aspect of the present invention is to detect the reagent of long-chain non-coding RNA in preparation diagnosis or prognosis tumor reagent
In application.In a specific embodiment, the tumour is gynecological tumor, preferably oophoroma；It is specific at another
Embodiment in, the long-chain non-coding RNA high expression in the ovarian cancer cell line that MEHP is handled；Further, institute
The long-chain non-coding RNA stated is selected from linc01233, linc00205, MIR193BHG, GAS5 and NOP14-AS1, it is preferred that
The long-chain non-coding RNA is linc00205；In another specific embodiment, the detection reagent is affiliated
Detection reagent known to the technical staff of field may be selected from but not limited to primer, probe, antibody.
Another aspect of the present invention is inhibitor the answering in preparation tumor of long-chain non-coding RNA
With.In a specific embodiment, affiliated tumour is gynecological tumor, preferably oophoroma；It is specific real at another
It applies in example, the long-chain non-coding RNA high expression in the ovarian cancer cell line that MEHP is handled；Further, described
Long-chain non-coding RNA is selected from linc01233, linc00205, MIR193BHG, GAS5 and NOP14-AS1, it is preferred that described
Long-chain non-coding RNA be linc00205；In another specific embodiment, the inhibitor is art technology
There is the reagent for inhibiting long-chain non-coding RNA known to personnel, may be selected from but not limited to small molecule compound, RNA interfering, resist
Body etc..In a specific embodiment, the inhibitor is siRNA, and further, the sequence of the siRNA is SEQ
Shown in ID NO:2.
Detailed description of the invention
Influence of Fig. 1 MEHP to SKOV3, A2780 cell migration and invasive ability.(A)-(B) at the MEHP of various concentration
The healing state of scratch after reason SKOV3, A2780 cell 24 hours, 48 hours.Cell 0.1%DMSO (control), 20nM,
The MEHP of 100nM and 500nM is handled.(C)-(D) cell is invaded after MEHP processing SKOV3, A2780 of various concentration
Situation and statistical chart.
Influence of Fig. 2 MEHP to SKOV3, A2780 cell epithelia mesenchymal transformation.(A) and (B) ovarian cancer cell line
SKOV3, A2780 use blank control, 0.1%DMSO (solvent control), 20nM, 100nM, 500nM concentration MEHP processing in advance
24 hours, the MEHP as the result is shown of detected by Western blot can lower the expression quantity of epithelial marker object ZO-1, while between up-regulation
The expression quantity of matter marker N-cad, Vimentin and transcription factor Slug, internal reference are GAPDH.(C) and (D) fluorescence microscope
It is intracellular compared with control group (0.1%DMSO) after lower observation 500nM concentration MEHP is handled SKOV3, A2780 cell 24 hours
The change in fluorescence situation of mesenchymal Vimentin.(E) and (F) ovarian cancer cell line SKOV3, A2780 use 0.1% in advance
DMSO (control), 500nM concentration MEHP are handled 24 hours, use real time fluorescence quantifying PCR method analysis ZO-1's and Slug
The expression of mRNA.
The change of ovarian cancer cell SKOV3 long-chain non-coding RNA expression quantity after Fig. 3 MEHP exposure.(A) and the volcano (C)
Figure, blue dot: the expression of lncRNA does not have difference in control group and experimental group；Green point: with control group phase
Than the lncRNA that expression is lowered in experimental group；Red point: compared with the control group, expression occurs in experimental group
The lncRNA of up-regulation.(B) and (D) gene thermal map, red: compared with the control group, expression quantity is lowered in experimental group
lncRNA；Green: compared with the control group, the lncRNA that expression raises in experimental group.(E) and (F) ovarian cancer cell
It is that SKOV3, A2780 use the MEHP of 0.1%DMSO (control), 500nM concentration to handle 24 hours in advance, it is fixed using real-time fluorescence
The expression of PCR method detection long-chain non-coding RNA is measured, the mRNA expression of GAPDH is used as internal reference, experimental data
It is presented using mean ± standard deviation form, it is for statistical analysis using two sample t-tests.
Effect of Fig. 4 linc00205 in ovarian cancer cell line migration, invasion and Epithelial and stromal conversion.(A) and (B)
After completing cell transfecting, using real time fluorescence quantifying PCR method detection siRNA ovarian cancer cell line SKOV3,
Poor efficiency is struck in A2780.(C) and (D) is after transfection targets the higher siRNA of linc00205 efficiency, uses 0.1%
The healing state of scratch after DMSO, 500nM concentration MEHP are handled SKOV3, A2780 cell 24 hours, 48 hours.(E) exist with (F)
Transfection targeting the higher siRNA of linc00205 efficiency after, using 0.1%DMSO, 500nM concentration MEHP processing SKOV3,
There is a situation where invade and statistical chart cell after A2780.After completing transfection, SKOV3, A2780 cell use 0.1% in advance
After the MEHP of DMSO (control) and 500nM is handled 24 hours, 18 are incubated in the cell transwell for being covered with substrate glue later
Hour, it is finally fixed, dyes and takes pictures, the counting of three pictures of each processing group selection is simultaneously for statistical analysis.(G) and
(H) transfection target the higher siRNA of linc00205 efficiency after, SKOV3, A2780 cell using 0.1%DMSO,
The MEHP of 500nM concentration is handled 24 hours.Detected by Western blot detects mesenchymal N-cad, Vimentin and transcription
The expressing quantity of factor S lug, the expressing quantity of GAPDH is as internal reference.Use two sample t-tests or single factor test variance point
It analyses for statistical analysis.
Specific embodiment
MATERIALS METHODS
1, abortion syndrome SKOV3 purchase is in Chinese Academy of Sciences's stem cell bank (China, Shanghai), with containing 10% tire ox
McCoy ' the s5A culture medium culture of serum；Abortion syndrome A2780 comes from the laboratory Zhejiang University Fan Hengyu, with containing
The DMEM culture medium culture of 10% fetal calf serum；All cells are cultivated in 37 DEG C, 5%CO2Incubator in.
2, phthalic acid list ethylhexyl (mono-2-Ethylhexyl phthalate, MEHP) purchase is in the U.S.
Sigma company, CAS 4376-20-9, molecular formula C16H22O4, molecular weight 278.34, DMSO dissolution packing, -20 DEG C are protected from light guarantor
It deposits.
3, cell culture: cell recovery, cell passage, cell cryopreservation are operation well known to those skilled in the art
The experiment protocol in technology or reference cell system source.
4, cell exposure experiment:
After cell passage, when planting plate culture to logarithmic growth phase, MEHP liquid storage is diluted to respectively with complete medium
The working concentration of 0nM, 20nM, 100nM and 500nM.According to the hole 6 orifice plates 2mL/；12 holes orifice plate 1mL/；96 orifice plate, 100 hole μ L/
Culture medium dosage, carries out contamination processing to cell by way of changing liquid.
5, cell proliferation experiment (CCK-8 method surveys cell viability)
1) inoculating cell: taking SKOV3, A2780 cell in logarithmic growth phase, will be adherent with the method for cell passage
Cell processing is uniform cell suspension, counted using cell counting board and adjusted cell concentration to 2 with complete medium ×
104A/mL, according to the volume of every 100 μ L cell suspension of hole by SKOV3, A2780 cell inoculation in 96 orifice plates.By 96 orifice plates
It is placed in 37 DEG C of incubators overnight.2) contaminate: after cell is adherent, with contain various concentration (10-8To 10-5M)DEHP、MEHP
Complete medium cell is carried out to change liquid, handle respectively for 24 hours, 48h and 72h.3) develop the color: after terminating contamination, every hole is added
10 μ L of CCK-8 reagent continues to be incubated for 1h in constant incubator.450nm excitation wavelength is selected, is surveyed using enzyme-linked immunosorbent assay instrument
The absorbance in each hole is measured, result is recorded.The ratio between absorbance by calculation processing sample and check sample indicates cell viability,
Every group of setting three parallel repetitions.
6, scratch Healing Experiments:
1) inoculating cell: SKOV3, A2780 cell of logarithmic growth phase, with the method for cell passage, by attached cell
It is processed into dispersion, uniform single cell suspension, adjustment concentration of cell suspension is counted, according to every hole cell quantity 4 × 105It is a to incite somebody to action
SKOV3, A2780 cell inoculation are in 6 orifice plates.6 orifice plates are put in and continue to cultivate in incubator.2) reach 100% patch to cell
After conjunction, by the way that with 200 μ L pipette tips of sterilizing, scratch-off surface keeps cell monolayer injured as far as possible uniformly, as the crow flies, obtain
Scratch.PBS buffer solution rinse three times, be added serum free medium, and carry out DEHP, MEHP contamination processing (0nM, 20nM,
100nM,500nM).It is placed in constant incubator and continues to cultivate 48h.3) according to 0h, for 24 hours, the point in time sampling of 48h take pictures, see
Examine the healing state of scratch.
7, Transwell Matrigel
1) gelatinization of Matrigel substrate is frozen overnight, pipette tips and 4 DEG C of eppendorf pipe pre-coolings.2) ice is beaten, Matrigel is placed
Substrate glue, serum free medium, pipette tips and eppendorf are managed.The cell transwell is put into the aperture of 24 orifice plates, small
Room edge number.Be vortexed the Matrigel for mixing and having thawed, and is diluted with the ratio of 1:40 with serum free medium, blows and beats several lower mixed
It is even to be placed on ice.The culture medium of every 100 μ L serum-free of hole, 2.5 μ L substrate glue, 1 hole of polygamy.After taking 100 μ l to dilute
Matrigel substrate glue is added in the cell transwell, avoids being added on side wall, not generate bubble.Set 1-2h in incubator.
3) cell (fusion degree 70-80%) to be processed is abandoned into culture medium, with the method for cell passage, attached cell is processed into list
Cell suspension is abandoned pancreatin after centrifugation, is resuspended with serum free medium.It counts and cell concentration is adjusted to 1.25 × 105A/mL,
(A2780 cell is 5 × 105A/mL).4) in 1) cell transwell Matrigel substrate glue sucked after being coated with it is extra
Liquid (serum free medium) takes 200 μ L cell suspensions (containing 2.5 × 10 immediately4A cell) cell is added, prevent basilar memebrane dry
It is dry.Poisonous substance is added, 600 μ L complete mediums are added in lower room, it is ensured that infiltration support membrane/poly- carbon ester film does not have bubble attachment, cultivates
12-16h is placed in case.5) 24 orifice plates for being placed with the cell transwell are taken out, 2 holes are selected in 24 orifice plates, 1mLPBS is added
Culture medium in cell is outwelled, is put into PBS buffer solution by buffer, and 1mLPBS is added in upper chamber, repeated washing 2 times, not stabbed
To film.6) 4% paraformaldehyde of 600 μ L is added in 24 holes, upper chamber is not added, and stands 5-20min.As step is slow with PBS in 5)
Fliud flushing is cleaned twice.7) 500 μ L Crystal Violet Dyes are added in 24 orifice plates, dye 5min.PBS buffer solution cleans room 2-3 up and down
It is secondary, upper cell 2-3 times of film is wiped with wet cotton swab, microscopically observation overall situation.8) after dry with microscope into
Row is taken pictures, and shoots three photos in case subsequent statistical.
8, protein blot experiment, " the Molecular Cloning: A Laboratory hand that RNA is extracted and reverse transcription experiment is published according to Cold SpringHarbor
Volume " operation.
9, transcript profile sequencing analysis
1) Quality Control of RNA and quantitative: monitoring RNA degradation and pollution on 1% Ago-Gel, measures the purity, dense of RNA
Degree and integrality carry out subsequent experimental after sample passes.2) for the library preparation of lncRNA sequencing: each sample draws 3 μ
GRNA is used as the raw material that RNA sample prepares.Firstly, the rRNA in removal RNA sample, and removed by ethanol precipitation
Residue without rRNA.Then, sequencing library is established using the RNA for eliminating rRNA.3) it clusters and is sequenced: using TruSeq PE
Cluster Kitv3-cBot-HS (Illumia) carries out the poly- of sample on cBot Cluster Generation System
Class.In fasciation at rear, library is sequenced on 4000 platform of Illumina Hiseq, and generates double ends reading of 150bp.
4) data analyze: cDNA library sequencing after, using FastQC tool check FASTQ format initial data (original reading) with
Carry out quality control.It is connected subsequence, repetitive sequence, reading and low-quality of the unknown nucleotide (" N ") greater than 10% by removal
Amount reading, generates the data to be analyzed of high quality from the original reading in each library.All subsequent analyses are all based on these
The data to be analyzed of high quality.After removal linking subsequence, low quality reading, we analyze the difference of experimental group and control group
Different expressing gene (DEG).It is analysed to data and is mapped to reference sequences Emsembl91.We calculate FPKM (fragments
Per kilo-base per million reads) it is horizontal to assess gene transcript expression, analysis of gene differential expression is completed,
Q value (p value of FDR adjustment) or the gene of p value < 0.05 are defined as difference expression gene as the candidate base further analyzed
Cause.5) functional analysis of difference expression gene: to difference table on http://www.heatmapper.ca/expression/
Thermal map analysis has been carried out up to gene (DEG).Upper GO, KEGG enrichment for reconciling down-regulated gene is analyzed by DAVID website making
(https: //david.ncifcrf.gov/).GO enrichment analysis is in bioprocess, the side such as molecular function and cell constituent
Face carries out respectively.
10, it statisticallys analyze:
All experimentss are in triplicate or more than three times.Meet the measurement data of normal distribution in data result with mean
± standard deviation indicates, and carries out significant difference analysis with t inspection or variance analysis.Use 5 data of GraphPad Prism
Processing software is for statistical analysis and maps, using value < 0.05 P as the standard of significant difference.
The influence of embodiment 1, nanomole dosage MEHP to abortion syndrome migration and invasive ability
We first probe into low dosage MEHP (0nM, 20nM, 100nM, 500nM) in vitro to ovarian cancer cell SKOV3,
The influence of A2780 migration and invasive ability.We have found that using the MEHP of 20nM, 100nM or 500nM concentration gradient
(metabolite of DEHP) is handled SKOV3, A2780 cell 24,48 hours, and the healing rate of scored area is compared with control group phase
Than significant increase has occurred, when being handled using 500nM concentration, healing rate variation is particularly evident.With scratch Healing Experiments
As a result consistent, compared with the control group, DEHP processing can not make the invasive ability generation conspicuousness of SKOV3, A2780 cell change
Become；And MEHP processing can dramatically increase the invasive ability of SKOV3, A2780 cell.Above-mentioned experimental result prompt, MEHP, DEHP
Hydrolysate, can but dramatically increase the invasive ability of ovarian cancer cell, and promote the transfer of ovarian cancer cell, MEHP's
Experimental phenomena is the most obvious when concentration for the treatment of is 500nM.The result is shown in Figure 1.
Epithelial and stromal conversion occurs for 2 MEHP of embodiment exposure induction ovarian cancer cell
Before the experimental results showed that the MEHP of low dosage can dramatically increase ovarian cancer cell invasive ability, promote ovum
The transfer of nest cancer cell.Tumour cell occur invasion and transfer the first step be EMT, we using MEHP (0nM, 20nM,
100nM and 500nM) after stimulation SKOV3, A2780 cell 24 hours, it is found that two plants of ovarian cancer cell lines have occurred EMT's
Process.As shown in Fig. 2, we observe the situation of change of EMT marker using detected by Western blot, MEHP exposure is so that epithelium
The expressing quantity of marker ZO-1 is lowered；Mesenchymal Vimentin, N-cad and associated transcription factor simultaneously
The expressing quantity of Slug is raised, wherein the variation of expressing quantity is the most obvious when being handled using 500nM concentration.
In addition to this, the method that we also use immunofluorescence observes the intracellular mesenchymal of ovarian cancer cell line SKOV3, A2780
The expression of Vimentin albumen handles cell using 500nM concentration MEHP as the result is shown in fluorescence microscopy microscopic observation
When, compared with the control group, green fluorescence increased significantly processing group, show that Vimentin expressing quantity increases；By glimmering in real time
The expression of the method detection EMT correlation marker mRNA level in-site of Fluorescent Quantitative PCR, using control group as reference, MEHP processing 24
After hour, the reduction of the mRNA expression generation conspicuousness of epithelial cell marker object ZO-1, and the relevant transcription factor of EMT
The mRNA expression of Slug produces the increase with statistical significance.The above experimental result by detected by Western blot,
Immunofluorescence technique and quantitative real-time PCR demonstrated in terms of protein level, mRNA level in-site MEHP exposure (500nM,
It can induce ovarian cancer cell line SKOV3, A2780 that EMT occurs for 24 hours).
The change of long-chain non-coding RNA expression quantity after 3 MEHP of embodiment exposure
We observe lncRNA expression after MEHP exposure.LncRNA of the p value less than 0.05 is defined as difference
Different expressing gene (DEGs).It is identical as the analysis method of mRNA, it has been found that compared with control group lncRNA expression quantity, MEHP
The change for having statistical significance occurs for the expression quantity that exposure group one shares 754 lncRNA, such as the volcano Fig. 3 figure and gene thermal map
Shown, the expression quantity of 695 lncRNA raises in 754 lncRNA, and the expression of 59 lncRNA generates downward.I
In discrepant 754 lncRNA of expression quantity that screen, include 39 certified lncRNA and 715 potential
lncRNA.We have chosen obtained confirmation 39 lncRNA it is further studied, probe into the lncRNA of these differential expressions
With the relationship of ovarian cancer cell line migration, invasive ability increase and EMT.As shown in the volcano Fig. 3 figure and gene thermal map, 39 differences
In the certified lncRNA of different expression, compared with the control group is shared after MEHP processing in 27 lncRNA expression quantity generations
It adjusts, the lncRNA of expression quantity decline has 12.In previous experiments result, after the MEHP exposure of 500nM concentration, ovarian cancer cell line
The migration of SKOV3, A2780, invasive ability increase, and EMT have occurred, and show that MEHP processing can promote ovarian cancer progression, because
In this sequencing result, compared to control group, the lncRNA that expression quantity raises in experimental group is potential rush tumour lncRNA.
It can be used as the target spot of oncotherapy due to promoting tumour lncRNA, there is potential therapeutic value, our seminars pay close attention to for a long time
Specific effect and mechanism of the lncRNA in terms of promoting tumor development, therefore next we study these potential rush
Whether tumour lncRNA influences the migration, invasion and the generation of EMT of Ovarian Cancer Cells SKOV3, A2780.
Firstly, we fasten the survey of verifying lncRNA with the method for real-time fluorescence quantitative PCR in two plants of ovarian cancer cells
Sequence result.As shown in figure 3, in SKOV3 cell, after the MEHP processing by 500nM concentration, what expression quantity raised
LncRNA has linc01233, linc00205, MIR193BHG, GAS5 and NOP14-AS1, wherein linc01233 with
The expression quantity up-regulation of linc00205 is the most significant, and up-regulation multiple has reached 1.5 times or more；And in A2780 cell, this five
LncRNA expression quantity is equally verified with the method for real-time fluorescence quantitative PCR.Experimental result is shown, in low dosage MEHP
After exposure, the lncRNA that two plants of ovarian cancer cell lines occur obviously to raise simultaneously is linc00205, shows that the pole linc00205 has
Certain effect may be played during ovarian cancer progress, increases migration, the invasive ability of tumour cell, and induces EMT
Generation.Therefore, we devise a series of experiment for linc00205, to verify our guess.
Effect of 4 linc00205 of embodiment in ovarian cancer cell line migration, invasion and Epithelial and stromal conversion
Firstly, we devise three siRNAs (siRNA) for linc00205, real-time fluorescence quantitative PCR is utilized
Method validation siRNA poor efficiency is struck for linc00205 in ovarian cancer cell line SKOV3, A2780.Described
Shown in the sequence following table of siRNA:
Experimental result is struck inefficient best small dry in two plants of ovarian cancer cell lines as shown in figure 4, after transfection 48 hours
Disturbing RNA is si-L1, therefore we select this siRNA of si-L1 to carry out subsequent experiment.Previous experimental result is
It proves, after the MEHP processing of 500nM concentration, the migration of ovarian cancer cell line SKOV3, A2780, invasive ability, which have occurred, has system
Meter learns the increase of meaning, and induces EMT；At the same time, after low dosage MEHP exposure, ovarian cancer cell line SKOV3, A2780's
The expression quantity of linc00205 is also raised.So we assume that low dosage MEHP exposure is by raising linc00205
The migration of expression quantity and then promotion ovarian cancer cell, invasive ability.In order to verify this guess, we are in ovarian cancer cell line
After the expression quantity for striking low linc00205 in SKOV3, A2780, migration, invasive ability and the EMT marker of tumour cell are observed
The change of expressing quantity.As shown in figure 4, in SKOV3 and A2780 cell, after the MEHP processing of 500nM concentration,
Linc00205 strikes low group compared with the control group, and the drop of conspicuousness all has occurred in the transfer ability and invasive ability of tumour cell
It is low, while linc00205 strikes the protein expression of mesenchymal N-cad, Vimentin and EMT transcription factor Slug in low group
Amount then has the reduction of conspicuousness compared with the control group.Above-mentioned experimental result can tentative confirmation, linc00205 can mediate low
Migration of Ovarian Cancer Cells, the increase of invasive ability and the generation of EMT of dosage MEHP exposure induction, strike low linc00205's
After expression quantity, the phenomenon that Migration of Ovarian Cancer Cells, the increase of invasive ability and EMT, takes a turn for the worse.
Our research illustrates low dosage environment incretion interferent MEHP from the angle of macroscopic view and regulates and controls to make in oophoroma
Signaling transduction networks in, the gene expression profile and the non-volume of long-chain of comprehensive analysis low dosage MEHP regulation ovarian cancer progress
The expression of code RNA, and above group credit analysis result preliminary identification linc00205 is combined to promote ovary in low dosage MEHP
The effect played in cancer progress provides certain enlightenment for subsequent research.
Above-described embodiment is only the preferred forms to show Research Thinking of the invention, anyone skilled in the art
The technical solution that can obviously obtain is disclosed according to the prior art each fall within the application want within de-protected invention.
Sequence table
<110>Zhejiang University
<120>long-chain non-coding RNA LINC00205 is preparing diagnosis of ovarian cancer reagent or is treating the application in ovarian cancer
<160> 4
<170> SIPOSequenceListing 1.0
<210> 3
<211> 7181
<212> DNA
<213>homo sapiens (Homo sapiens)
<400> 3
gccctcctga ccgcagcggc tagaggttcc attgcagacc cggaggccgt ggctgtggtt 60
cgcggcggtg ctgtcgcggg cgccctggcg cagcccacgc aggggctcct gagggtccgc 120
gaggccggga ggtccggggg tcgggaggtc ccggggtcgg gaagtcggtg gaccctgcag 180
gccagtgggg aggggagaca taagacatca gtcagtacat gtgaggtgtg cattggattg 240
atccagaaag tcaggacgac tccaagtgga aaggcctcca gagacaggag acagtaaaag 300
aaatggcctc tctcctctgt cagagtggag tcccgagctc ccagaaatgc cacatgatgg 360
acaaacgctt gggggaaaaa aaaaaaaaag gagacctcag tcggacacaa aagcaggagc 420
tttaaaagaa aaaataagag ataagttgaa cttcaccaaa aaataagcgt ttctggtctt 480
tgaaagacac ttaaaatgaa aaggcaagcc atcgatgggg aaaatactca gaataatgga 540
tctgacagag ggctgtatct agaatatatg aagatatttt aaaatccagt aatttgacat 600
aaagatggta gccagcctga cacacaaagg aacccgaatg gccaatggac aggaagggaa 660
cccgtatggc cgatggacag aaagggaagt gaacgtgaac atctcattgg ggatgccgtt 720
atcaccactg gaatagtcaa aaacatggac accaagtatt ggtgggtgag tggaatggcg 780
ggaacccaca tacacggtcg atgggagctg caccctttgg aaatcatttc acagtttgtg 840
ctccatcctg gccaaaccca cagctggagt ctttccaaaa acagtgacgg gagtttccac 900
cagggagtgc ctcaaaccat cccgagatgg ggctggttgg gattccaggg agaggcactc 960
gttgccaggg tgatccgtcc agagcacttc ctaggggatc tggcataagg agggctgcag 1020
tgtatcctca ggacagatag gacagatagt gtatctttgg gacagacggc aagatgggga 1080
ttccacccag gtctgtctgc agcgagttga tatgagagtt taaggaattt ggaccagggc 1140
tgctttgttt cagggttttg ggcaatgacc taaacacctt tatcagtgcc tgggaatgct 1200
caaggcccag cttgagttca ggcctgcagg gaaaacctgc aactggccgg gctgcagagt 1260
ggtcagggca cggaaagcca gaagctgggg acacacctgc tgtatgatcc agccgttcgt 1320
ctttacctgg gagaaatggc accacctggc tgaacataac ttcttacact cccatgagtc 1380
ctcctggaca ggtgctgtga cctcgtgcag ccttggggac cctgacactc ccacggacag 1440
gccaaggggt ttgcctgggc cctgtgggca cagagcccct tgagatgggt ttccctatgc 1500
agccccccac cccccaccgg ggacccatgg cactgcagcc ctcacccccc ctctagggac 1560
ccacaacact gcagccctcg cccccctccg gggtcccaca gcactgcagc cctcgccccc 1620
catcggggac ccacagcact gcagccacgc ccaggccacc gcctccaaac acagggccgc 1680
tgctgtttct gtgaacagat acttcttgca gatgtcaatg gttaatggat ggggaggtga 1740
ccgcccaagc agaagccgac cctcttcatg aaggggccac aggtcacccc gaagcagaaa 1800
tccatagaac agccaaggcc acagcggaac cgagcaggcc acgcctctgc ctctgggccg 1860
ctcagggcca ggcctccctg accccactgg ctctattgtg aggactcagg gtggagctct 1920
gctgggctca gtggccttca cagccggggt ccacagaccc gggtcctacg ggcagtactg 1980
tgctcaccta gaggccacgg tccatgccct gtctccagca ggcccaagtc acttgtccat 2040
ggcgaaggcc cggccttgct tcaggccccg ggcgcgctcc ccctgctccc cctgcccacc 2100
gtttcttccc aggtgacaga ggcgggagag caggcgagcc acgtgccggg cgccgcagca 2160
gggggcactc caggctggcg cccctctgcc tctccgtggg ctctgacctt tcttctccct 2220
gcctgggcag cctccttcag gttggggagt cttttgttga cccctggatt aaagtcctaa 2280
tcaggaatga cccggaagag gtcttatgag gctttcctca agagaagaaa atctgtccct 2340
gagtatcagg aagtggcccc tttccctgca ccgcagtctt ctgtgaccag cggctcacac 2400
agcgaaggag gggctggcgg cccccacagg ccactgcccc gaggccgcca cgagagggca 2460
ggagagccct tcctgggagc tctgtgccac ggggaaacgc agccccgcca agcacagcat 2520
ggatgtttcc agcagggaat gaagagagag gccccagcac atgagagaag agcacactgg 2580
gcggcccctg tcggagcctc tcctgtcccc agcgacccct gagagcaggc cctgggcctc 2640
tgcggcccct cccggagcca ctcttcaact gctgacctgc tcccagctct tctgtgcccc 2700
gcccagctcc ctgcaaaacc tcacctgagg ggaaggaggc cctgtttggg ctcagacgca 2760
gcaggtgcag cttgtggtcc tggggccaca cctgttagag cccatccttc tacccctgct 2820
gggccctgag tgtcgtccgt ccccacagaa ctcagcaggg tcaggtctgg gcactccagg 2880
ccgccagccc cctgagtgag ccctgggccg gaggtagttg tgggtcacag gcaccccagc 2940
cagaacaccc acagtgggta gatgtgtggg gaccggatgt gggtcctctc ctgagagacg 3000
tgcagatggg gagaggctgg gcagttctca gcacagctgg agcatctacc tgtcaccacc 3060
tcggagcctc ctggccccgt gggggcggcc ctgggagcag ggtcggcagt gaagagcaga 3120
gaaaggcagt tggggatgtt gccactgtcc cccgagacca ccctgccatg gagacgaggg 3180
agctgtccct tcgcggaagg gggctggcca gcaagaagga cagagagtgg actggccgag 3240
gaccgctgag ctcaggaccc aaggaggact cttctaggag acgagagagc gaacgccagg 3300
gaccctgtgc aggcctgctc ctccgtttgc aaggtgagtt accaggttca cgtgtttgga 3360
gtttctggac tcattgcgga gttccacccc tgcacgttgc ggttccccag taatcaaatc 3420
ctggcttttg tgcctggaag tgcacaggga ggggacaact ttgtgagtca gtggcagggc 3480
agggagttct ggttctccag agccagaggc cgtgctcaga aggatttctt agcaggagcc 3540
ttggggcccc cagtcaacac ttccctacgg acagcctggc cagctctgtg cacggagcag 3600
gcgcccgagg gtcccaggtc accaagtgac caagtcgtga aggcgcccag ggtttcctgg 3660
gggtgcgctg atcccaagga agccacgtgt ggtcagcatg ggggagggga ccagcgcccc 3720
ggggggcctg cagcacagca gggcctgccc tcctgggtga gactggccgg tgcctgtggg 3780
gatctggggg gctacagtca gggctctgtg ctcccgaggg ccacgccagc ccacctgccc 3840
tggaacagaa cccgaggctt ctgcctaggg gagtacctgg gcacctgcct cctgtgctgc 3900
cctggacaca tcccagcggc tgcacatagg ggaggcacag cctgggctca gggccagggt 3960
ccactctgtg gggatactag acccgggggt gacaatgcca gctcagaact tccccccaca 4020
cctgggctcc tccagcctgg cctttcctgg ggaagggagg gcctgtcctt cctcagcact 4080
gtgggaggga ggcaaggcct gcggttgaag cgtccccaac acggggctcc aggagagaaa 4140
gcaccacatc tcagggaatg aaaacagaga cggggccgcc cagtgcactc ggtggctcgg 4200
aaatcattaa agaatgttct gagcccccga ttttggctgt aaaagggact ggccggcttg 4260
tgaccgctcc cctgtctgtg ccttgagacg ggagtgttca gcgttggggg cagctttccc 4320
tccaagagga gcttcacaaa catccgctct ctgcggggcc gcctctccgt ggcctggggc 4380
cgctgtcgga ggaaggctct ccagctgccg tcatctggga aacgtggggg gcgagcaggg 4440
gtcatggatg gggctcactg gggactgtga gaatctgtcc cgcaggactt tctgggatgg 4500
aaacgctggc agaggtgaag cctgcgtcat gtgcttcact gaacccggct gcttatttat 4560
gttcggaggg ctggtttcaa ggactcctcg tctccctctc cagtgatagc gtcagcggaa 4620
atgcagacgg ggacggggct gctgggtttc ctccctggaa tgaagcacag ccggaggttt 4680
tgctgattca ccagcaggcc ctgaccgctg agttcagggt aacaaatcca catggatcct 4740
gagcccgcat agctccctgg gcctcagcat aaacatcgta aacctccggg ccctggcagt 4800
gtctgtttca tcccctccag agcaagtgca agtgtgagat ttagagattc taagagggga 4860
agggccagag gtcttgaaaa cgaccttcat tcgttcattc attcgagaag tttgcgaggg 4920
cctccgtgtc ctgcgtttag tggggcctag gacgcagatg tgaagggcgc tgctctcagg 4980
agcagacaga ggtggggatg aatcagtaca cgcccacctc gggcccacag gggaggatgc 5040
gcggagctgt gggtgtgggg aggagacccc tctgcgtcat gctgacggtg tcggcagagt 5100
cgccagcttc tgcaggagcg gtacctgtgg gttcttctgt gacttgttta accgcatctt 5160
ttgcccagta gttagtcttt tcctgttggg acaccatgtt ggtagtttgg aaatggtttc 5220
ttccatccat tgcctgcctt ttagctttgt cgatggtgtt ctgttgtaaa ttttggtgca 5280
cgtttaatgt gaacaatggt tatgagacga gtgccatgag ttcctgtgtg cctgtcaccc 5340
agcccggcca caagaggtgc tgggggcagt gtccacaccc ccctttctta ggacgcctga 5400
gtctcagatg tgacttatag ggtatttctt atggcaagac ggttaaaaca aacttcagcg 5460
tctcgtctgt ccttctatgg ctgtggcttc tgatgttcta atggcgttct cgtcagccgg 5520
ggctgagaac aaaataacat agactgtggg gcttaaacag cagaaactta cttcccacgg 5580
ttctggaggt tgggagtctt ggatcaccgt gtagcatggt caggttcctg gtgagggtgg 5640
gattcctggc taacgtaacg aaggctccct ctcctgatac cgtgtcactg ggggtgaggc 5700
ttcaacacag gaattttggg gggacacatc agcattcact ccatcacagg tggttagccc 5760
tttaatccgc gggaattttg tttggggttg tgtgagatac gggtctaacg ttttcttttt 5820
caaatacgta gccagttgtc acatcattta ttgaaaaagg aatcttttct ccaccgactg 5880
acatgaaatg ctaccatcat cgtaaataaa attcccgtaa atacttgctg tctctgctgt 5940
ctcagtcctg actcacgggc tgagttctct ttctgcacag tagcactggc attaactgtg 6000
acagctttac agcaggctcc ctccccgagg ccgttcagaa gcattcctca gcgggtccta 6060
cacgtttcct ctcccatgtc aagtttagaa gcagtgtcaa gacccacagc agtcctgcgg 6120
gagttttaag ggatgcacgg agtttatggg gacagtttgg aaaattgaca ttcatgtgac 6180
ttagagtcct actacttgaa aatggattcc agctctcaac gaatttagag ctttggcaaa 6240
atttttaaga tttctttgat gtccgatgtg ctcatttctt ggtttgttct tgagtatttt 6300
gtggattttt atgaaatcca caaagttttt gttataatga atgggacact ttcccataaa 6360
atgttgtaat tctgtattgc tgttttagta aacactgttg attgatgtat attgatgtta 6420
cacttggtca cttgtaatag tttgtccgtt cattattttg aactttttag gtaaacagtc 6480
atataattat gcaaataatt atagttgtgt ctctgccttt ctaatattta tactttgtgt 6540
atattatcat gttggccagg actcaagcgt ctttctcttg tttctgacta atgcgaatga 6600
ttctaatgca ggggtttcca aactggtggc cggggggcca aatccagcca atggtctctt 6660
cttgtaaata aagttttatt ggaacacagt tacacacatt tttctacata ttgtctgatg 6720
gctactgtca cgccacagca atgctgttaa atagtccaga cagaggcggt attgcccgaa 6780
aaacctagaa tattcaccat ctgagctttt acgggaaaat ttgctaatat ctgttctcat 6840
gcattaaata caatgtttgt tacaggttaa ggaagtttct gactattttt agctttctga 6900
atatcttgtg gttgtgtgtg ctttaaaatt aggactaaat attaaattta ccagttgctt 6960
gttaggggcc tatcttttga gatgcccaaa gtttcccttt tttagtctct tcatgtagtg 7020
agttgtacta acagattctc taatgttgaa ccgtctttgc tttccggaga tagactttac 7080
ttgctcctgg tggattggat tctgtttgtt aatactttta tttttgggta attacatccc 7140
tattataaat aatatgtcag catcaaaaaa aaaaaaaaaa a 7181
<210> 2
<211> 21
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gccguuauca ccacuggaat t 21
<210> 3
<211> 21
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gcaguacugu gcucaccuat t 21
<210> 4
<211> 21
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ccuguuagag cccauccuut t 21
Claims (10)
1. long-chain non-coding RNA is preparing the purposes in diagnosing tumor product, which is characterized in that the long-chain non-coding RNA
It is that incretion interferent (EDC) induces highly expressed long-chain non-coding RNA, the tumour is gynecological tumor, preferably ovary
Cancer.
2. purposes described in claim 1, wherein the EDC is phthalic acid list ethylhexyl (MEHP).
3. the described in any item purposes of claim 1-2, which is characterized in that the long-chain non-coding RNA is linc00205.
4. the described in any item purposes of claim 1-3, which is characterized in that the sequence of the long-chain non-coding RNA is SEQ
Segment in sequence shown in ID NO:1, the segment being capable of specificity characterization SEQ ID NO:1 expression；Or with SEQ ID
NO:1 has 99% homology and derives from the RNA sequence of the mankind；Or the long-chain non-coding RNA is SEQ ID NO:1
Shown in sequence.
5. the reagent of the expression of long-chain non-coding RNA described in any one of detection claim 1-4 is preparing diagnosing tumor
Purposes in product, the tumour are gynecological tumor, preferably oophoroma.
6. application of the inhibitor of long-chain non-coding RNA in preparation tumor, which is characterized in that the long-chain is non-
Coding RNA is that incretion interferent (EDC) induces highly expressed long-chain non-coding RNA, it is preferred that the EDC is adjacent benzene two
Formic acid list ethylhexyl (MEHP).
7. purposes as claimed in claim 6, which is characterized in that the tumour is gynecological tumor, preferably oophoroma.
8. the described in any item purposes of claim 6-7, which is characterized in that the long-chain non-coding RNA is linc00205.
9. the described in any item purposes of claim 6-8, it is characterised in that the inhibitor is RNA interfering.
10. the described in any item purposes of claim 6-9, it is characterised in that the inhibitor is RNA interfering, the interference
The sequence of RNA is as shown in SEQ ID NO:2.
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| CN113278696A (en) * | 2021-05-14 | 2021-08-20 | 浙江大学医学院附属妇产科医院 | Molecular marker RAD51B-AS1 and application thereof |
| CN115737813A (en) * | 2021-09-03 | 2023-03-07 | 深圳大学总医院 | Application of LINC00205 inhibitor in preparation of medicine for treating liver cancer |
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